Mitochondria are the major source of cellular energy (ATP), as well as critical mediators of widespread functions such as cellular redox balance, apoptosis, and metabolic flux. The organelles play an especially important role in the maintenance of cardiac homeostasis; their inability to generate ATP following impairment due to ischemic damage has been directly linked to organ failure. Methods to quantify mitochondrial content are limited to low throughput immunoassays, measurement of mitochondrial DNA, or relative quantification by untargeted mass spectrometry. Here, we present a high throughput, reproducible and quantitative mass spectrometry multiple reaction monitoring based assay of 37 proteins critical to central carbon chain metabolism and overall mitochondrial function termed 'MitoPlex'. We coupled this protein multiplex with a parallel analysis of the central carbon chain metabolites (219 metabolite assay) extracted in tandem from the same sample, be it cells or tissue. In tests of its biological applicability in cells and tissues, "MitoPlex plus metabolites" indicated profound effects of HMG-CoA Reductase inhibition (e.g., statin treatment) on mitochondria of i) differentiating C2C12 skeletal myoblasts, as well as a clear opposite trend of statins to promote mitochondrial protein expression and metabolism in heart and liver, while suppressing mitochondrial protein and ii) aspects of metabolism in the skeletal muscle obtained from C57Bl6 mice. Our results not only reveal new insights into the metabolic effect of statins in skeletal muscle, but present a new high throughput, reliable MS-based tool to study mitochondrial dynamics in both cell culture and in vivo models.

中文翻译：

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MitoPlex：一种有针对性的多反应监测测定，用于定量一组精选的线粒体蛋白。

线粒体是细胞能量 (ATP) 的主要来源，也是细胞氧化还原平衡、细胞凋亡和代谢流等广泛功能的关键介质。细胞器在维持心脏稳态方面发挥着特别重要的作用。由于缺血性损伤导致它们无法产生 ATP，这与器官衰竭直接相关。定量线粒体含量的方法仅限于低通量免疫测定、线粒体 DNA 测量或通过非靶向质谱法进行相对定量。在这里，我们提出了一种基于高通量、可重复和定量质谱多反应监测的检测方法，对中央碳链代谢和整体线粒体功能至关重要的 37 种蛋白质进行检测，称为“MitoPlex”。我们将这种蛋白质多重分析与从同一样品（无论是细胞还是组织）中串联提取的中心碳链代谢物（219 代谢物测定）进行平行分析结合起来。在细胞和组织中的生物学适用性测试中，“MitoPlex 加代谢物”表明 HMG-CoA 还原酶抑制（例如他汀类药物治疗）对 i) 分化 C2C12 骨骼肌成肌细胞的线粒体产生深远影响，以及与他汀类药物明显相反的趋势促进心脏和肝脏中线粒体蛋白的表达和代谢，同时抑制从 C57Bl6 小鼠获得的骨骼肌中的线粒体蛋白和 ii) 代谢方面。我们的结果不仅揭示了他汀类药物在骨骼肌中代谢作用的新见解，而且呈现出新的高通量，